The present invention relates to the nucleotide sequence of the gene encoding the horse cytokine GM-CSF, to expression vectors containing it, and to its use as adjuvant in equine vaccination and as nonspecific immunity stimulant.
The documents cited here are incorporated by reference into the present application. (All documents cited herein, and all documents cited in documents cited herein are hereby incorporated herein by reference).
The first discovery of a granulocyte-macrophage colony-stimulating factor (GM-CSF) dates from 1977 (Burgess A.W. et al. J. Biol. Chem. 1977, 252, 1998-2003). It is the murine GM-CSF, purified from mouse lung culture supernatants.
The biological activities of GM-CSF have been demonstrated by the work carried out on the murine and human GM-CSFs (Clark S. C. et al. Science 1987, 230, 1229; Grant S. M. et al. Drugs 1992, 53, 516).
GM-CSF has many physiological roles (Dy M. in xe2x80x9cLes cytokinesxe2x80x9d Cavaillon J.-M., 1996, ed. Masson, Paris, France, 43-56). In particular, GM-CSF stimulates the production, the development and the formation of colonies of granulocytes, macrophages, eosinophils and megakaryocytes. GM-CSF induces in particular a macrophagic cytotoxocity, stimulates antibody-dependent cytotoxic activity (ADCC) and the recruitment of leukocytes at the level of the sites of inflammation.
The GM-CSFs from various animal species have already been identified.
The sizes of the nucleotide sequences encoding the known GM-CSFs from various species vary from 381 to 432 nucleotides. The human and murine nucleotide sequences have a degree of homology of 69%. The degree of homology is 54% at the level of the amino acid sequence (Cantrell M. A. et al. Proc. Natl. Acad. Sci. USA 1985, 82, 6250-6254). However, this homology does not allow any cross-activity between the two human and murine species (Metcalf D. et al. Blood 1986, 67, 37-45).
The administration of heterologous GM-CSF, that is to say obtained from a species other than the one treated, does not make it possible to obtain an optimum adjuvant effect, in particular a stimulation of the activity of the haematopoietic cells and a substantial increase in the immune response.
Up until now, it has not been possible to identify the equine GM-CSF. Yet this cytokine is of great interest for therapeutic and vaccinal applications for use in horses.
The applicant has succeeded in isolating and sequencing the equine GM-CSF gene. This gene was isolated after polymerase chain reaction (PCR) with the aid of the oligonucleotides described in the examples.
The equine GM-CSF gene has a size of 432 nucleotides (SEQ ID No. 8 and FIG. 1) and encodes a protein of 144 amino acids (SEQ ID No. 9 and FIG. 1). The protein encoded by this gene exhibits a homology of at least 75% with the GM-CSF polypeptide sequences of other animal species.
The subject of the present invention is therefore an isolated DNA fragment encoding equine GM-CSF, e.g. a fragment comprising SEQ ID No. 8. Its subject is also the DNA fragment having or consisting essentially of this sequence.
The subject of the present invention is also an isolated DNA fragment encoding the amino acid sequence SEQ ID No. 9.
The invention covers the equivalent nucleotide sequences of equine or synthetic origin, that is to say the nucleotide sequences encoding a protein of equivalent functionality and specificity in horses. The nucleotide sequences which differ by the degeneracy of the genetic code will of course be included. In particular, DNA sequences having an homology equal or greater than 90%, particularly than 92%, preferably than 95% with SEQ ID No. 8, are equivalent sequences.
An object is also DNA fragments comprising such a nucleotide sequence encoding the equine GM-CSF, e.g. according to SEQ ID No. 8 or a sequence encoding the amino acid sequence SEQ ID No. 9, this nucleotide sequence being associated, in the form of a fusion, with the nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen. The DNA fragment then does not comprise a stop codon between the sequence encoding GM-CSF and the associated immunogen encoding sequence. For instance, referring to SEQ ID No. 8, the coding sequence inserted ends at nucleotide 432, and does not include the stop codon.
The subject of the present invention is also the isolated equine GM-CSF protein or polypeptide, e.g. that encoded by the nucleotide sequence SEQ ID No. 8 or by the equivalent of the latter as defined above.
The subject of the present invention is also the equine GM-CSF protein having the amino acid sequence SEQ ID No. 9.
The equine GM-CSF protein has a size of 144 amino acids. However, the present invention also comprises the proteins, protein fragments and polypeptides of equine origin or which are synthetic, having a size greater or equal than or less than these 144 amino acids, as well as the recombinant proteins (having one or more substitutions, deletions or additions) and the fusion proteins, as long as their biological activity (for the part which is common to GM-CSF) is substantially equivalent to that of the natural equine GM-CSF protein in vivo in horses and their species-specificity is not modified. Are encompassed as equivalents any of the amino acid sequences encoded by any of the equivalent nucleotide sequences as defined above.
The subject of the present invention is also a pure preparation of equine GM-CSF protein.
The subject of the present invention is also the expression vectors containing, as insert, any of the above defined DNA fragments or nucleotide sequences, in particular the equine GM-CSF gene (SEQ ID No. 8) or an equivalent thereof as defined above, as well as any of the nucleotide sequences encoding any of the above defined amino acid sequences. Also, the vector may further comprise a nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen, which can be or not associated under the form of a fusion as described above.
The nucleotide sequence may be inserted into conventional in vitro expression systems of viral origin, such as Baculovirus, in particular propagated on insect cells, or cells of prokaryotic origin (for example Escherichia coli) or eukaryotic origin, in particular yeasts, especially Saccharomiyces cerevisiae, mammalian eukaryotic cells, especially hamster cells (for example hamster ovary cells or CHO) and horse cells. The invention therefore also covers expression systems transformed by a sequence according to the invention, the equine GM-CSF proteins thus produced and their use as adjuvant for vaccine and nonspecific immunity stimulant.
Preferably, the sequence according to the invention is introduced into in vivo expression vectors under conditions allowing the expression, in horses, of a functional equine GM-CSF protein, and possibly a nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen. These expression vectors may be plasmids, viral vectors, such as poxviruses, for example the vaccinia virus, avipoxviruses (canarypox, fowlpox), including the species-specific poxviruses (swinepox, raccoonpox and camelpox), adenoviruses and herpesviruses, such as the equine herpesviruses.
The term plasmid is intended to cover any DNA transcription unit in the form of a polynucleotide sequence comprising the sequence of the equine GM-CSF gene and the elements necessary for its expression in vivo. The circular plasmid form, supercoiled or otherwise, is preferred. The linear form also falls within the scope of this invention.
Each plasmid comprises a promoter capable of ensuring, in the host cells, the expression of the gene inserted under its control. It is in general a strong eukaryotic promoter and in particular a cytomegalovirus early promoter CMV-IE, of human or murine origin, or optionally of other origin such as rat or guinea pig. More generally, the promoter is either of viral origin or of cellular origin. As a viral promoter other than CMV-IE, there may be mentioned the SV40 virus early or late promoter or the Rous Sarcoma virus LTR promoter. It may also be a promoter from the virus from which the gene is derived, for example the promoter specific to the gene. As cellular promoter, there may be mentioned the promoter of a cytoskeleton gene, such as for example the desmin promoter, or alternatively the actin promoter. When several genes are present in the same plasmid, they may be provided in the same transcription unit or in two different units.
The plasmids may also comprise other transcription regulating elements such as, for example, stabilizing sequences of the intron type, preferably intron II of the rabbit bxc3xaata-globin gene (van Ooyen et al. Science, 1979, 206: 337-344), signal sequence of the protein encoded by the tissue plasminogen activator gene (tPA; Montgomery et al. Cell. Mol. Biol. 1997, 43: 285-292), and the polyadenylation signal (polyA), in particular of the bovine growth hormone (bGH) gene (U.S. Pat. No. 5,122,458) or of the rabbit beta-globin gene.
The invention also covers the immunogenic compositions and the vaccines comprising the equine GM-CSF protein according to the invention, and at least one immunogenic or vaccinal preparation of equine pathogen, and a veterinarily acceptable excipient or vehicle. The notion of immunogenic preparation covers here any preparation capable, once administered to horses, of inducing an immune response directed against the equine pathogen considered, a response which is increased by the presence of the GM-CSF protein. It is preferably a vaccinal preparation capable of inducing an effective protection or a degree of protection against this pathogen, a degree of protection which is increased here by the presence of the equine GM-CSF protein. The immunogenic and vaccinal preparations intended in the invention cover all the known types, such as inactivated, attenuated live, subunit and recombinant (using an in vivo expression vector, in particular of viral or plasmid origin). As was seen above, the GM-CSF protein may be added as such to the immunogenic or vaccinal preparation to form, in the presence of veterinarily acceptable excipient or vehicle, an immunogenic composition or a vaccine ready to be administered. It is also possible to envisage combining the GM-CSF protein with a prolonged-release system designed to gradually release the protein.
According to a more advantageous mode of the invention, it is however preferable to express the GM-CSF protein in vivo using an in vivo expression vector as described above. In this case, it is also preferable that the immunogenic or vaccinal preparation is also of the recombinant type, based on the use of an in vivo expression vector, of the same type or of a different type. It is also possible to envisage using the same in vivo expression vector, comprising and expressing at least one equine pathogen immunogen and the equine GM-CSF protein.
The advantages of the use of GM-CSF during vaccinations are in particular the reduction in the dose of immunogen or of vector or DNA used. Furthermore, in some animals which do not respond when administered with a customary vaccine, the use of GM-CSF allows the stimulation of the immune response and its increase up to a protective level.
The present invention therefore preferably covers the immunogenic compositions and the vaccines comprising:
an in vivo expression vector containing a nucleotide sequence encoding an equine GM-CSF under conditions allowing the expression, in horses, of a functional equine GM-CSF protein,
at least one in vivo expression vector containing at least one nucleotide sequence encoding at least one equine immunogen, it being understood that this vector or some or all of these vectors (when there are several vectors encoding various immunogens) may also constitute the GM-CSF vector (say the vector comprises at least the GM-CSF sequence and an immunogene sequence), and
a veterinarily acceptable vehicle or excipient.
According to a preferred mode of the invention, the invention covers the immunogenic compositions and the vaccines of the DNA type, comprising a plasmid encoding and expressing the equine GM-CSF according to the invention and at least one other plasmid encoding and expressing an equine immunogen or an immunologically active fragment derived therefrom. Examples of plasmid constructs which contain an equine immunogen and which can be used in the invention are given in patent application WO-A-9803198. The invention also covers the DNA vaccines comprising a plasmid encoding and expressing simultaneously the equine GM-CSF and at least one equine immunogen.
The invention covers all the equine pathogens. There may be mentioned more particularly equine herpesvirus type 1 or type 4 (and preferably the invention provides for combination of both types), equine influenza virus, tetanus, Borrelia burgdorferi, Eastern, Western and Venezuelan equine encephalites, rabies virus. For the subunit vaccines and the recombinant vaccines, the equine immunogens are preferably selected from the group comprising the glycoproteins gB, gC and gD of the equine herpesvirus type 1 or type 4, the haemagglutinin (HA) and the nucleoprotein (NP) of the equine influenza virus, the C subunit fragment of the tetanus toxin, the Borrelia burgdorferi OspA protein, the Eastern, Western and Venezuelan equine encephalites E2 and C genes, the rabies virus G gene.
The subject of the present invention is also nonspecific immunity stimulating compositions, that is to say which can be used as a general immunity stimulant in horses. These compositions are administered in the presence or in the absence of a declared pathology, in general independently of any vaccine, in order to reinforce the immune defenses in horses. These compositions comprise GM-CSF according to the invention, in all the forms described above, protein or recombinant, preferably recombinant (in vivo viral or plasmid expression vector), and a veterinarily acceptable excipient or vehicle. The characteristics of these vectors have already been described.
The nonspecific stimulating compositions and the immunogenic compositions and the vaccines according to the invention may also comprise one or more immunity adjuvants, in particular selected from those normally used in equine vaccination against the pathogens (valencies) considered. The stimulating compositions and the immunogenic compositions and conventional vaccines (inactivated, attenuated live, subunit) may thus comprise, as conventional adjuvant, compounds of the carbomer or aluminum hydroxide type, or may be formulated in the form of an oil-in-water emulsion. For the stimulating compositions and the immunogenic compositions and recombinant vaccines based on a viral expression vector, there may be mentioned the oil-in-water emulsions.
According to a preferred mode of the invention, for the stimulating compositions of the plasmid type and the immunogenic compositions and vaccines of the plasmid type, the plasmid encoding and expressing the equine GM-CSF, the plasmid encoding and expressing GM-CSF and at least one equine immunogen, as well as the mixtures of plasmids containing the latter and at least one plasmid encoding an equine immunogen, may be advantageously formulated in a novel fashion with a cationic lipid containing a quaternary ammonium salt,of formula: 
in which R1 is a saturated or unsaturated linear aliphatic radical having 12 to 18 carbon atoms, R2 is another aliphatic radical containing 2 or 3 carbon atoms, and X a hydroxyl or amine group It is preferably the DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium; WO-A-9634109), preferably coupled with a neutral lipid, DOPE (dioleoylphosphatidylethanolamine), to form preferably DMRIE-DOPE. Preferably, the recombinant vector mixture with this adjuvant is made immediately before use and preferably, before its administration to the animal, the mixture thus produced is allowed to form a complex, for example over a period ranging from 10 to 60 minutes, in particular of the order of 30 minutes.
When DOPE is present, the DMRIE:DOPE molar ratio preferably ranges from 95:5 to 5:95, more particularly 1:1.
The plasmid:DMRIE or DMRIE-DOPE adjuvant weight ratio may range in particular from 50:1 to 1:10, in particular from 10:1 to 1:5, preferably from 1:1 to 1:2.
According to another advantageous mode of the invention, for the stimulating compositions of the recombinant type and the immunogenic compositions and vaccines of the recombinant type (viral vector or plasmid), it is possible to use, as adjuvant, polymers of acrylic or methacrylic acid or copolymers of maleic anhydride and of alkenyl derivative. The polymers of acrylic or methacrylic acid crosslinked in particular with polyalkenyl ethers of sugars or of polyalcohols are preferred. These compounds are known by the term carbomer (Pharmeuropa vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 (incorporated by reference) describing such acrylic polymers crosslinked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced with unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopolxe2x96xa1 (BF Goodrich, Ohio, USA) are particularly appropriate. They are crosslinked with an allyl saccharose or with allylpentaerythritol. Among them, there may be mentioned Carbopol(copyright) 974P, 934P and 971P.
Among the copolymers of maleic anhydride and of an alkenyl derivative, the EMAs(copyright) (Monsanto) are preferred which are copolymers of maleic anhydride and ethylene, linear or crosslinked, for example crosslinked with divinyl ether. Reference may be made to J. Fields et al., Nature, 186: 778-780, Jun. 4, 1960 (incorporated by reference). From the point of view of their structure, the polymers of acrylic or methacrylic acid and the EMAs(copyright) preferably consist of basic units of the following formula: 
in which:
R1 and R2, which are identical or different, represent H or CH3 
x=0 or 1, preferably x=1
y=1 or 2, with x+y=2
For the EMAs(copyright), x=0 and y=2. For the carbomers, x=y=1.
The dissolution of these polymers in water leads to an acidic solution which will be neutralized, preferably to physiological pH, to give the adjuvant solution into which the actual vaccine will be incorporated. The carboxyl groups of the polymer are then partly in COO form.
Preferably, a solution of carbomer or of EMA(copyright) is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the required quantity (in order to obtain the desired final concentration), or a substantial part thereof, of water loaded with NaCl, preferably physiological saline (NaCl 9 g/l), in one or more portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pH will be used as it is to mix with the immunogenic or vaccinal preparation, in particular stored in lyophilized, liquid or frozen form.
The polymer concentration in the final vaccine composition will be 0.01% to 2% W/V, more particularly 0.06 to 1% W/V, preferably 0.1 to 0.6% W/V.
Another object of the invention is a method of immune stimulation and/or immunisation and/or vaccination of equine species, wherein a stimulating, immunogenic or vaccine composition according to the invention is administered to an animal from equine species, in particular a horse. Administration is preferably done via parenteral route, such as intramuscular,intradermal or sucutaneous route. One or more administrations can be done. In particular, in case of vaccination, administration is done each time the vaccine is administered.
The quantity of DNA used in the stimulating compositions and the immunogenic compositions and vaccines according to the present invention is between about 10 xcexcg and about 2000 xcexcg, and preferably between about 50 xcexcg and about 1000 xcexcg, for a given plasmid. Persons skilled in the art will have the competence necessary to precisely define the effective dose of DNA to be used for each therapeutic or vaccination protocol.
If a live vector is used, doses may be between 104 and 1010 Pfu (plaque forming unit) preferably between 106 and 108 pfu.
For a composition containing GM-CSF protein, doses may be from 1 xcexcg to 5 mg, preferably from 50 xcexcg to 1 mg.
The dose volumes may be in particular between 0.5 and 5 ml, preferably between 2 and 3 ml.